The effects of metoclopramide on the central nervous system (CNS) in patients suggest substantial brain distribution. Previous data suggest that metoclopramide brain kinetics may nonetheless be controlled by ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier. We used 11 C-metoclopramide PET imaging to elucidate the kinetic impact of transporter function on metoclopramide exposure to the brain. Methods: 11 C-metoclopramide transport by P-glycoprotein (P-gp; ABCB1) and the breast cancer resistance protein (BCRP; ABCG2) was tested using uptake assays in cells overexpressing P-gp and BCRP. 11 C-metoclopramide brain kinetics were compared using PET in rats (n 5 4-5) in the absence and presence of a pharmacologic dose of metoclopramide (3 mg/kg), with or without P-gp inhibition using intravenous tariquidar (8 mg/kg). The 11 C-metoclopramide brain distribution (V T based on Logan plot analysis) and brain kinetics (2-tissue-compartment model) were characterized with either a measured or an imaged-derived input function. Plasma and brain radiometabolites were studied using radio-high-performance liquid chromatography analysis. Results: 11 C-metoclopramide transport was selective for P-gp over BCRP. Pharmacologic dose did not affect baseline 11 C-metoclopramide brain kinetics (V T 5 2.28 ± 0.32 and 2.04 ± 0.19 mL⋅cm −3 using microdose and pharmacologic dose, respectively). Tariquidar significantly enhanced microdose 11 C-metoclopramide V T (7.80 ± 1.43 mL⋅cm −3 ) with a 4.4-fold increase in K 1 (influx rate constant) and a 2.3-fold increase in binding potential (k 3 /k 4 ) in the 2-tissue-compartment model. In the pharmacologic situation, P-gp inhibition significantly increased metoclopramide brain distribution (V T 5 6.28 ± 0.48 mL⋅cm −3 ) with a 2.0-fold increase in K 1 and a 2.2-fold decrease in k 2 (efflux rate), with no significant impact on binding potential. In this situation, only parent 11 C-metoclopramide could be detected in the brains of P-gp-inhibited rats. Conclusion: 11 C-metoclopramide benefits from favorable pharmacokinetic properties that offer reliable quantification of P-gp function at the blood-brain barrier in a pharmacologic situation. Using metoclopramide as a model of CNS drug, we demonstrated that P-gp function not only reduces influx but also mediates the efflux from the brain back to the blood compartment, with additional impact on brain distribution. This PET-based strategy of P-gp function investigation may provide new insight on the contribution of P-gp to the variability of response to CNS drugs between patients.

Translocator protein (TSPO) is expressed at a low level in healthy brain and is upregulated during inflammatory processes that may occur in neurodegenerative diseases. Thus, TSPO may be a suitable in vivo indicator of neurodegeneration. Here, we quantified the 18 F-DPA-714 radioligand in healthy TSPO-genotyped volunteers and developed a method to eliminate the need for invasive arterial blood sampling. Methods: Ten controls (7 high-affinity binders [HABs] and 3 mixed-affinity binders [MABs]) underwent 18 F-DPA-714 PET with arterial and venous sampling. 18 F-DPA-714 binding was quantified with a metabolite-corrected arterial plasma input function, using the 1-and 2-tissue-compartment models (TCMs) as well as the Logan analysis to estimate total volume distribution (V T ) in the regions of interest. Alternative quantification methods were tested, including tissue-to-plasma ratio or population-based input function approaches normalized by late time points of arterial or venous samples. Results: The distribution pattern of 18 F-DPA-714 was consistent with the known distribution of TSPO in humans, with the thalamus displaying the highest binding and the cerebellum the lowest. The 2-TCM best described the regional kinetics of 18 F-DPA-714 in the brain, with good identifiability (percentage coefficient of variation , 5%). For each region of interest, V T was 47.6% ± 6.3% higher in HABs than in MABs, and estimates from the 2-TCM and the Logan analyses were highly correlated. Equilibrium was reached at 60 min after injection. V T calculated with alternative methods using arterial samples was strongly and significantly correlated with that calculated by the 2-TCM. Replacement of arterial with venous sampling in these methods led to a significant but lower correlation. Conclusion: Genotyping of subjects is a prerequisite for a reliable quantification of 18 F-DPA-714 PET images. The 2-TCM and the Logan analyses are accurate methods to estimate 18 F-DPA-714 V T in the human brain of both HAB and MAB individuals. Population-based input function and tissue-to-plasma ratio with a single arterial sample are promising alternatives to classic arterial plasma input function. Substitution with venous samples is promising but still requires methodologic improvements.

Increasing evidence suggests that neuroinflammation is active in Parkinson disease (PD) and contributes to neurodegeneration. This process can be studied in vivo with PET and radioligands targeting TSPO, upregulated in activated microglia. Initial PET studies investigating microglial activation in PD with the [ 11 C]-PK11195 have provided inconclusive results. Here we assess the presence and distribution of neuroinflammatory response in PD patients using [ 18 F]-DPA714 and to correlate imaging biomarkers to dopamine transporter imaging and clinical status. Methods: PD patients (n = 24, Hoehn and Yahr I-III) and 28 healthy controls were scanned with [ 18 F]-DPA714 and [ 11 C]-PE2I and analyzed. They were all genotyped for TSPO polymorphism. Regional binding parameters were estimated (reference Logan graphical approach with supervised cluster analysis). Impact of TSPO genotype was analyzed using Wilcoxon signed-rank test. Differences between groups were investigated using a two-way ANOVA and Tukey post hoc tests. Results: PD patients showed significantly higher [ 18 F]-DPA714 binding compared to healthy controls bilaterally in the midbrain (p < 0.001), the frontal cortex (p = 0.001), and the putamen contralateral to the more clinically affected hemibody (p = 0.038). Microglial activation in these regions did not correlate with the severity of motor symptoms, disease duration nor putaminal [ 11 C]-PE2I uptake. However, there was a trend toward a correlation between cortical TSPO binding and disease duration (p = 0.015 uncorrected, p = 0.07 after Bonferroni correction). Conclusion: [ 18 F]-DPA714 binding confirmed that there is a specific topographic pattern of microglial activation in the nigro-striatal pathway and the frontal cortex of PD patients.

Abstract. Glyburide (glibenclamide, GLB) is a widely prescribed antidiabetic with potential beneficial effects in central nervous system injury and diseases. In vitro studies show that GLB is a substrate of organic anion transporting polypeptide (OATP) and ATP-binding cassette (ABC) transporter families, which may influence GLB distribution and pharmacokinetics in vivo. In the present study, we used [11 C] GLB positron emission tomography (PET) imaging to non-invasively observe the distribution of GLB at a non-saturating tracer dose in baboons. The role of OATP and P-glycoprotein (P-gp) in [11 C]GLB whole-body distribution, plasma kinetics, and metabolism was assessed using the OATP inhibitor rifampicin and the dual OATP/P-gp inhibitor cyclosporine. Finally, we used in situ brain perfusion in mice to pinpoint the effect of ABC transporters on GLB transport at the blood-brain barrier (BBB). PET revealed the critical role of OATP on liver [ 11 C]GLB uptake and its subsequent impact on [11 C]GLB metabolism and plasma clearance. OATP-mediated uptake also occurred in the myocardium and kidney parenchyma but not the brain. The inhibition of P-gp in addition to OATP did not further influence [11 C] GLB tissue and plasma kinetics. At the BBB, the inhibition of both P-gp and breast cancer resistance protein (BCRP) was necessary to demonstrate the role of ABC transporters in limiting GLB brain uptake. This study demonstrates that GLB distribution, metabolism, and elimination are greatly dependent on OATP activity, the first step in GLB hepatic clearance. Conversely, P-gp, BCRP, and probably multidrug resistance protein 4 work in synergy to limit GLB brain uptake.

[(18)F]DPA-714 binds to TSPO with high specificity in the primate brain under normal conditions and in the QA model. This tracer provides a sensitive tool for assessing neuroinflammation in the human brain.

]DPA-714 were investigated in rats, baboons, and humans. Whole-body PET experiments showed a high uptake of radioactivity in the kidneys, heart, liver, and gallbladder. The liver was a major route of elimination of [ 18 F]DPA-714, and urine was a route of excretion for radiometabolites. In rat and baboon plasma, high-performance liquid chromatography (HPLC) metabolic profiles showed three major radiometabolites accounting for 85% and 89% of total radioactivity at 120 minutes after injection, respectively. Rat microsomal incubations and analyses by liquid chromatography-mass spectrometry (LC-MS) identified seven metabolites, characterized as O-deethyl, hydroxyl, and N-deethyl derivatives of nonradioactive DPA-714, two of them having the same retention times than those detected in rat and baboon plasma. The third plasma radiometabolite was suggested to be a carboxylic acid compound that accounted for 15% of the rat brain radioactivity.

The interaction between rat and human liver cytochromes P450 with a series of lysergic acid derivatives and ergopeptide alkaloids was studied by difference visible spectroscopy. Ergopeptides, like bromocriptine, ergocryptine and dihydroergotamine, strongly interacted with rat liver microsomes with the appearance of a difference spectrum which is characteristic of their binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P450s 3A in the microsomes and was at its maximum in dexamethasone-treated rat microsomes. All the ergopeptides studied exhibited a high affinity for rat P450s 3A (K-around 1 pM), although lysergic acid derivatives not bearing the tripeptide moiety failed to give significant interactions with these P450s. A cyclic azatripeptide exhibiting a structure very similar to that of the tripeptide moiety of ergopeptides also interacted with P450s 3A with appearance of an intense type I difference spectrum. Very similar results were observed with two allelic forms of human liver P450 3A4, P450 NF25 and P450 hPCN1, produced in yeast. In both cases all the ergopeptides studied showed high affinities for the P450s (K? 0.6-2.2 pM) and an intense shift from the low-spin to the high-spin state upon substrate binding (60-100% spin shift). Lysergic acid derivatives not bearing the tripeptide group of ergopeptides also completely failed to interact with P450s 3A4.Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P450 3A, were found to be particularly active for the hydroxylation of bromocriptine, which occurs at the level of its tripeptide moiety. Human liver microsomes as well as P450 NF25 and P450 hPCNl also exhibited a high activity for bromocriptine hydroxylation at this level.These results show that ergopeptides exhibit a particularly high affinity for P450s of the 3A subfamily. The tripeptide moiety of ergopeptides is essential for their recognition by P450s 3A and binds at a site close to P450 heme, producing type-I difference spectra. Accordingly, at least one of the studied ergopeptides, bromocriptine, is hydroxylated by P450s 3A at the proline ring of the cyclopeptide moiety. As cyclosporine is known to be a good substrate of P450s 3A, these results suggest that P450s 3A may be especially prone in a general manner to recognize and oxidize peptides or pseudopeptides.Cytochrome P450 enzymes constitute a superfamily of heme-thiolate proteins that catalyse the primary oxidation of a wide variety of natural endogenous substrates like steroids, fatty acids, prostaglandins, leukotrienes and lipid hydroperoxides. They also play an important role in the metabolism of exogenous compounds like drugs, procarcinogens, solvents, anesthetics and environmental pollutants. Their broad substrate specificity is now well understood on the basis of enzyme multiplicity (Gonzalez, 1992). More than 220 P450s have been sequenced and characterized; they have been classified on the basis of primary amino acid sequence similarity (Nelson et...

Human liver P450 NF25 (CYP3A4) had been previously expressed in Succhuromyces cerevisiue using the inducible GALIO-CYCl promoter and the phosphoglycerate kinase gene terminator [Renaud, J. p., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194,. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72,463 -4721 increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25 -iron-metabolite complexes. %fold, g-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6@-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b, always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25 -iron-metabolite complex. A P450 Fe(I1)-(RNO) complex was obtained upon oxidation of Nhydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase. These results show that specific activities of yeast-expressed P450 NF25 may be artificially low, owing to limiting amounts of the associated microsomal redox proteins and emphasize the importance of controlling the amounts of the different components of the monooxygenase complex in order to optimize these catalytic activities, especially when the expression system is to be used for demonstrating metabolic capacities towards new substrates.